SUMMARY: The Preclinical Development and Clinical Monitoring Facility (PDCMF) projects have developed from transplantation protocols developed within ETIB. Peripheral blood, marrow aspirates, and tumor and CGVHD tissue biopsies from all ongoing ETIB protocols are processed and preserved by the PDCMF. We have evaluated lymphocyte subsets, cytokine content, T cell receptor repertoire diversity, and gene expression to support research studies of clinical protocols. All data are incorporated into protocol-specific spreadsheets, linking samples to protocol arms and transplant time points, and are accessible by branch clinicians over secure NIH networks. The work of this core is supported by close collaborative relationships with the Cell Processing Service of DTM, for support of microarray analysis and development of clinical products; the ETIB Flow Cytometry Facility, for support of sorting of clinical products for research endpoints; and the laboratory of Ronald Gress, for technical support in RNA and DNA isolation and quantitative assays. PDCMF supports studies of lymphocyte reconstitution following ETIB transplant trials that include: (1) non-myeloablative reduced intensity allogeneic transplant for lymphoma using sibling and matched-unrelated donors, (2) autologous transplantation for myeloma and for systemic autoimmunity, and (3) myeloablative transplant for acute leukemias (04-C-0095, 04-C-0055, 07-C-0195, 08-C-0088, 11-C-0016, 11-C-0136; PIs Daniel Fowler, Steven Pavletic, Ronald Gress, Kirsten Williams and Christopher Kanakry (all ETIB)). In the past year, these immune monitoring studies have contributed to reports on allogeneic HSCT in lymphoma and renal carcinoma involving novel lymphodepletion regimens and utilization of adoptive transfer of T-RAPA cells (Fowler et al, Clin Cancer Res, 2015). We also support ongoing studies of lineage-specific immune reconstitution in patients transplanted for monogenic immune deficiencies (such as GATA2 or DOCK8 (09-C-0096, 10-C-0174, PI: Dennis Hickstein) and primary immune deficiencies (16-C-0003, PI: Jennifer Kanakry (ETIB)) by monitoring repopulation of deficient lineages after allogeneic transplant (Grossman et al, Biol Blood Marrow Transpl, 2014) and by working with the ETIB Flow Cytometry Facility (William Telford) to assess subset-specific donor chimerism. This past year we have analyzed immune reconstitution in a matched unrelated donor allogeneic transplant trial (07-C-0195: PI Steven Pavletic) comparing two distinct regimens of GVHD prophylaxis, one utilizing antibody-mediated depletion of donor lymphocytes following transplant, the second using immune suppressive agents. We determined that the lympho-depleting treatment resulted in severe and protracted reduction in T cell numbers for the first year, as compared to that in patients with immune suppression alone. Naive T cell populations, in particular, were severely reduced in the lymphodepleted arm, resulting in significant deficits in naive cell frequency and numbers at 6 and 12 months after transplant, that were only rectified by 24 months. Consistent with this deficit, we determined that the overall T cell receptor repertoire diversity in the lympho-depleted arm was significantly reduced throughout the first year. The lymphocyte repopulation differences between the two arms correlated with significant increases in early viral infections and relapse in the lymphocyte-depleted arm, and with significant increases in chronic GVHD (CGVHD) in the immune suppressive arm. We also identified a 10 fold difference between these two transplant arms in the median ratio of Treg cells/microliter to either naive CD4 or naive CD8 cells/microliter at 6 months, a time point prior to onset of CGVHD in most patients; these findings are consistent with evidence of a key role for naive T cells in initiation of CGVHD, and with the importance of the level of Treg cells in controlling CGVHD. CGVHD is the principle cause of non-relapsemorbidity and mortality after allogeneic transplantation, and has been a major focus for research in the PDCMF core. We have supported the efforts of the multidisciplinary CGVHD clinical team in an ongoing natural history protocol of CGVHD (P.I. Steven Pavletic: 04-C-0281) by characterizing immunologic changes of CGVHD. Furthermore, we have supported therapeutic trials for CGVHD patient populations by supporting biospecimen preservation (both blood and tissues) and by performing assays: (1) defining the role of Th2 lineage T cells and leukotrienes in bronchiolitis obliterans (BOS), a pulmonary complication of CGVHD (08-C-0097: P. I. Ronald Gress and Kirsten Williams); (2) characterizing Th17 and Treg populations in a trial of Imatinib, a TGFbeta signaling inhibitor, in sclerotic cutantaneous CGVHD (Dermatology and Pediatric Branch, protocol 08-C-0148, P.I. Edward Cowen and Kristin Baird) (Baird et al, Biol Blood Marrow Transpl, 2015); (3) assessing lymphocyte populations and cytokine production in a trial of a high-potency steroid administered as a mouthwash in oral CGVHD (12-C-0068; P.I. Steven Pavletic/Jacqueline Mays); (4) characterizing changes in effector and regulatory T cells in a trial of Pomalidomide (12-C-0197; P.I. Steven Pavletic) as a therapy against fibrosis in CGVHD patients. This year, we support (5) immune reconstitution studies in a new allogeneneic transplantation trial using Keratinocyte Growth Factor (Pallifermin) to reduce the incidence of GVHD (15-C-0067: PI Steven Pavletic/Lauren Curtis), and (6) pharmacodynamics and biospecimen collections for a trial of an oral neutrophil elastase inhibitor, to reduce neutrophil activation in BOS (16-C-0060: PI Steven Pavletic/Annie Im). (7) Two additional trials will be opening shortly, involving Ponesimod, an inhibitor of lymphocyte trafficking into tissues (15-C-0171: PI Steven Pavletic/Lauren Curtis) and Baracitinib, an inhibitor of JAK1/JAK2 mediated immune activation (PI Steven Pavletic/Annie Im). As secondary research endpoints for trials 4 - 7, we are assessing the effects of these therapies on a series of CGVHD biomarkers that we have developed through our analyses of CGVHD. We have proposed that Interferon (IFN)-induced factors recruit Th1/Tc1 effector T and regulatory cells into CGVHD targeted tissues (Imanguli et al, Blood 2009; Imanguli et al Leukemia 2014). We profiled gene expression of circulating monocytes in CGVHD patients, to use these cells as in situ reporter cells for systemic cytokine patterns. Based on microarray analysis and confirmed by multiplex RNA assessments, we determined that pathways induced by IFN were consistently upregulated across a broad spectrum of CGVHD patients, both those with severe inflammatory infiltrates in tissue and those with widespread sclerotic involvement. IFN-inducible genes, including ones specifically induced by type I IFN, were upregulated in parallel at onset of CGVHD, and were reduced during treatment and after resolution of CGVHD symptoms. Furthermore, we found a consistent upregulation of receptor genes from the innate immune pathways which can induce IFNa production, consistent with a self-reinforcing inflammatory process sustaining CGVHD. Based on these analyses we have established flow cytometry panels to assess Th1/Tc1 effector and Treg cell levels in blood, ELISA panels to assess IFN inducible chemokines, and custom probe sets to assess the gene expression profiles we have found to characterize both newly initiated and active, established CGVHD. This extensive experience in immunologic characterization of CGVHD has also supported our participation in the CGVHD consensus report on CGVHD biomarker studies (Paczesny et al, Biol Blood Marrow Transpl, 2015).